Isfahan University of Medical SciencesResearch in Pharmaceutical Sciences1735-53621120071011Non-radioactive single-strand conformation polymorphism (SSCP) analysis of relatively long PCR products81420101210<font face="Times New Roman"><span style="font-size: 11pt">The SSCP technique is based on the appearance of new &ldquo;refolding&rdquo; conformations during electro-phoresis due to mutation. In order to develop a simple, non-radioactive SSCP analysis method so that it can reliably detect single nucleotide changes in PCR products up to 500 bp in length, </span><span style="font-size: 11pt">extensive optimisation trials were performed</span><span style="font-size: 11pt">. </span><span style="font-size: 11pt">The best separation of SSCP bands of PCR products up to 500 bp in length was obtained with 14.5 to 15.5% polyacrylamide gels that had been run in the cold room at 15 V/cm for 65 to 70 hours. After the pre-run, 5 </span></font><span style="font-size: 11pt; font-family: Symbol"><span>m</span></span><span style="font-size: 11pt"><font face="Times New Roman">l of PCR product was mixed with 10 </font></span><span style="font-size: 11pt; font-family: Symbol"><span>m</span></span><span style="font-size: 11pt"><font face="Times New Roman">l of denaturing-loading dye and the mixture was heated to 94 </font></span><span style="font-size: 11pt; font-family: Symbol"><span>&deg;</span></span><span style="font-size: 11pt"><font face="Times New Roman">C for 10 min. Total mixture volume of the 15 </font></span><span style="font-size: 11pt; font-family: Symbol"><span>m</span></span><font face="Times New Roman"><span style="font-size: 11pt">l was loaded into the well without quenching. After electrophoresis, the gels were stained with SYBR<sup>&reg;</sup> Gold nucleic acid stain for 30-40 minutes and photographed under UV light using a SYBR<sup>&reg;</sup> gel stain photographic filter. </span><span style="font-size: 11pt">This method alongside with confirmatory sequencing has been utilised to identify three novel mutations in the 5</span></font><span style="font-size: 11pt; font-family: Symbol"><span>&cent;</span></span><span style="font-size: 11pt"><font face="Times New Roman">-regulatory region of the human <em>CYP3A4</em> gene. The final results were very satisfactory and the optimised method was able to reliably reveal new mutations in amplified DNA from blood samples. In fact the non-radioactive SSCP method developed here seems to be robust enough to analyse PCR products up to 500 bp long.</font></span>http://rps.mui.ac.ir/index.php/jrps/article/view/6http://rps.mui.ac.ir/index.php/jrps/article/download/6/4